There is a growing medical need for safe and effective antifungal agents that stems from the rapidly increasing population of immune-compromised patients. Currently available therapeutic agents act on targets that are also found in mammalian cells. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and morphogenesis. The principal mechanism by which yeast cells detect and respond to wall stress is a signaling pathway mediated by a family of cell surface sensors, a small GTPase (Rho1), protein kinase C (Pkc1), and a MAP kinase cascade, although additional pathways also contribute to the structural integrity of the wall. The study of yeast cell wall integrity is likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The specific aims of this project are: 1) To understand the function of the Hcs77 and Mid2 cell surface sensors for cell wall integrity signaling. Genetic and two-hybrid analyses will be used to test a model for the interaction of these sensors with the Rom1 and Rom2 guanosine nucleotide exchange factors for Rho1. If the model provides to be incorrect, three unbiased genetic and biochemical approaches will be taken to identify the signaling components with which these sensors interact. Finally, a model for these sensors as probes of the cell wall will be tested by examining the effect on signaling of length mutations in the extracellular domain of Hcs77. 2) To identify signaling components that function on a second branch of the Pkc-activated signaling pathway. Pkc1 has been proposed to regulate a bifurcated pathway with the MAP kinase cascade on one branch. A genetic screen for defect additivity with a component of the MAP kinase cascade will be used to identify signaling components that function on a second branch of this pathway. 3) To identify signals that activate the Ypk1 protein kinase, and the output of its signaling. Ypk1 and its redundant homologue, Ypk2, serve a function in the maintenance of cell wall integrity, and have been connected recently to phosphoinositide metabolism at the plasma membrane. Ypk1 protein kinase activity will be used as an assay to identify conditions that activate signaling through this molecule. The effect of Ypk1 activity on plasma membrane phosphoinositide levels will be examined in ypk1/2 mutants and under conditions of Ypk1 activation. 4) To understand the function of the Ypk1/Stt4 complex. Ypk1 resides in complex with Stt4-PI-4-kinase. Moreover, ypk1/2 mutants are suppressed by over expression of the Mss4 pI-5-kinase. These results implicate Ypk1/2 in the regulation of phosphoinositides at the plasma membrane. The level at which this regulation takes place will be explored.